Laccase are copper oxidases and are found in large quantities in several white rot fungi that are involved in lignin metabolism. Fungal laccases have boundless biotechnological functions across the globe like the decolouration and detoxification of industrial effluent, bleaching of pulp, phenolic elimination from wines, in preparation of biosensors in detergents blocking dye transfer- functions. Laccase showed vast variety of substrates due to this ability they can enhance different types of industrial mechanism such as methylation, demethylation, polymerization, mineralization of pollutants like hydrocarbons. White rot fungus is efficient for the production of laccase using agro-waste as substrate.
In this research white rot fungus was isolated from stock cultures of Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan and the organism was maintained on Tien& Kirk media slants and petri plates. Solid state fermentation technique was used using basal fermentation medium and agro waste rice bran was used as substrate for the production of laccase.Proximate analysis was performed of the substrate rice bran to analyse crude protein, fat, ash, moisture and fibre content. The fermentation was performed at room temperature and flasks were placed on orbital shaker at 100 rpm and 30˚C.
Enzyme activity was checked using (ABTS) as substrate at 420nm, for every 24 hour to observe the maximum enzyme production. Fermentation parameters like substrate concentration, incubation period, pH, temperature and nitrogen source (corn steep liquor and ammonium sulphate) were optimized. The concentration of substrate optimized for rice bran was 7.5g/100ml and optimum production of 6.11 IU/ml of enzyme was observed. The optimum day for the production of enzyme was day 7 and the amount of enzyme produced was 6.91 IU/ml. The optimum pH and temperature were 4 and 40˚C respectively, and the amounts of enzyme produced were 7.48 IU/ml and 7.96 IU/ml respectively. Two nitrogen sources optimized were maize steep liquor 1ml and ammonium sulphate 0.2 g, and the enzyme produced was recorded 7.67 IU/ml and 9.41 IU/ml respectively. Large scale fermentation batch of one litter was carried out under the optimized conditions and the enzyme produced was 9730 IU/L. Triplicates of each parameter were prepared.
The enzyme was purified using the purification techniques like ammonium sulphate precipitation, then by dialysis excess salt was removed, and then gel filtration was performed to collect different fractions on the basis of size of molecules and molecular mass of the laccase was analysed by SDS-PAGE. The size of the protein was found to be 70kDa. Characterization of laccase was performed in terms of optimum pH, temperature and in response to inducers and inhibitors. The optimum pH and temperature of the purified enzyme was 6 and 40˚C respectively. The inducer copper sulphate enhanced the activity of enzyme up to 9.7 U/ml and then inhibitors EDTA and 2-merceptoethanol reduced the activity level up to 4.17IU/ml and 3.98 IU/ml respectively. To study and analyse the effects of optimization parameters Pearson correlation, descriptive statistics and one way Anova were used.
The optimum production of laccase was achieved using agro waste rice bran. The enzyme produced was economical and it can provide effective solutions for bioremediation of hazardous compounds and pollutants.